Selective molecular transport through the protein shell of a bacterial microcompartment organelle

Chowdhury, Chiranjit; Chun, Sunny; Pang, A.H.; Sawaya, M.R.; Sinha, Sharmistha; Yeates, Todd O.; Bobik, Thomas A.

doi:10.1073/pnas.1423672112

Cited by 128 publications

(204 citation statements)

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“…Thus, studies of PduL support the model that both internal cofactor recycling and transport across the shell are required for the proper function of the Pdu MCP. Furthermore, recent in vitro studies demonstrated that purified Pdu MCPs restrict the influx of 1,2-PD but do not measurably impair the inward movement of HS-CoA and NAD ϩ (47). This strongly supports the idea of specific routes of cofactor entry into the Pdu MCP.…”

Section: Figmentioning

confidence: 53%

“…The function of the Pdu MCP requires that the enzymes encapsulated within be provided with a steady supply of their required substrates and cofactors. On the basis of crystallographic studies, it was proposed that the central pores seen in BMC-domain shell proteins provide specific conduits for metabolites (31)(32)(33)(34)(35)46), and recent studies showed that the PduA shell protein forms a selective pore tailored to allow the influx of 1,2-PD while restricting the efflux of propionaldehyde (47). Moreover, recent studies showed that enzymatic cofactors can be regenerated internally within MCPs to maintain steady cofactor supplies.…”

Section: Discussionmentioning

confidence: 99%

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The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

et al. 2015

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In Salmonella enterica, 1,2-propanediol (1,2-PD) utilization (Pdu) is mediated by a bacterial microcompartment (MCP). The Pdu MCP consists of a multiprotein shell that encapsulates enzymes and cofactors for 1,2-PD catabolism, and its role is to sequester a reactive intermediate (propionaldehyde) to minimize cellular toxicity and DNA damage. For the Pdu MCP to function, the enzymes encapsulated within must be provided with a steady supply of substrates and cofactors. In the present study, Western blotting assays were used to demonstrate that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that the N-terminal 20-residue-long peptide of PduL is necessary and sufficient for targeting PduL and enhanced green fluorescent protein (eGFP) to the lumen of the Pdu MCP. We present the results of genetic tests that indicate that PduL plays a role in the recycling of coenzyme A internally within the Pdu MCP. However, the results indicate that some coenzyme A recycling occurs externally to the Pdu MCP. Hence, our results support a model in which a steady supply of coenzyme A is provided to MCP lumen enzymes by internal recycling by PduL as well as by the movement of coenzyme A across the shell by an unknown mechanism. These studies expand our understanding of the Pdu MCP, which has been linked to enteric pathogenesis and which provides a possible basis for the development of intracellular bioreactors for use in biotechnology. IMPORTANCEBacterial MCPs are widespread organelles that play important roles in pathogenesis and global carbon fixation. Here we show that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that PduL plays a key role in cofactor homeostasis by recycling coenzyme A internally within the Pdu MCP. Further, we identify a potential N-terminal targeting sequence using a bioinformatic approach and show that this short sequence extension is necessary and sufficient for directing PduL as well as heterologous proteins to the lumen of the Pdu MCP. These findings expand our general understanding of bacterial MCP assembly and cofactor homeostasis. Bacterial microcompartments (MCPs) are a diverse family of subcellular proteinaceous organelles used to optimize metabolic pathways that have toxic or volatile intermediates (1, 2). MCPs are polyhedral in shape and about 100 to 150 nm in size and consist of a multiprotein shell that encapsulates sequentially acting enzymes of specific metabolic pathways. Overall, they are composed of several thousand protein subunits of 10 to 20 different types and may exceed a gigadalton in mass. Genomic analyses indicate that MCPs are produced by about 20% of bacteria and that there are seven or more different types (which function in diverse metabolic processes) as well as numerous subtypes (3-5). The first MCP identified was the carboxysome (6). This MCP functions to optimize carbon fixation, and it has been estimated that 25% of CO 2 fixation on Earth occurs within carboxysomes (2). Two other MCPs that have been studied are used for...

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Section: Figmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 99%

The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

et al. 2015

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“…However, recent structures of carboxysome shell proteins offer a potential mechanism for differential permeability of CO 2 and HCO 3 − : The pores of shell proteins typically carry positive charge, which might increase the rate of HCO 3 − transit relative to CO 2 (12,49). Indeed, recent experimental evidence suggests that protein compartments can be selectively permeable (50,51). Intuitively, it seems that a very high HCO 3 − permeability and a very low CO 2 permeability would be best for the efficient operation of the CCM.…”

Section: S Elongatus Cyotosolic Ph Is Within the Optimal Range For Ccmmentioning

confidence: 99%

pH determines the energetic efficiency of the cyanobacterial CO ₂ concentrating mechanism

Mangan

Flamholz

Hood

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

181

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Many carbon-fixing bacteria rely on a CO 2 concentrating mechanism (CCM) to elevate the CO 2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O 2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines two features: active transport of inorganic carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochemical intuition with a quantitative description of the system. To this end, we have developed a mathematical model of the CCM to analyze its energetic costs and the inherent intertwining of physiology and pH. We find that intracellular pH greatly affects the cost of inorganic carbon accumulation. At low pH the inorganic carbon pool contains more of the highly cellpermeable H 2 CO 3 , necessitating a substantial expenditure of energy on transport to maintain internal inorganic carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO 2 retention in the carboxysome is necessary, whereas selective uptake of HCO 3 − into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quantitatively consistent with cyanobacterial physiology, emphasizing that pH cannot be neglected when describing biological systems interacting with inorganic carbon pools.carbon fixation | RuBisCO | cyanobacteria | inorganic carbon | systems biology C yanobacteria and many other autotrophs use a CO 2 concentrating mechanism (CCM) to increase the cellular pool of inorganic carbon and facilitate the Calvin-Benson-Bassham (CBB) cycle (1). Specifically, the CCM functions to supply CO 2 to ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the primary carboxylating enzyme of the CBB cycle. High levels of CO 2 are essential to cyanobacterial metabolism because RuBisCO has relatively slow carboxylation kinetics and is promiscuous, catalyzing an off-pathway reaction with O 2 called oxygenation (2-4).RuBisCO oxygenation produces 2-phosphoglycolate (2PG), which is not part of the CBB cycle and must be recycled. Recycling 2PG through photorespiratory pathways is costly, consuming reduced carbon and energy resources (5, 6). The problem of RuBisCO's limited specificity is all the more pronounced because there is ≈ 20 times more O 2 than CO 2 in aqueous solutions equilibrated with present-day atmosphere (SI Appendix, Fig. S1). CCMs overcome these problems by concentrating CO 2 near RuBisCO, favorably increasing the ratio of CO 2 to O 2 . High concentrations of CO 2 maximize the rate of carboxylation and competitively inhibit oxygenation. Indeed, it is widel...

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“…Prior studies showed that Salmonella mutants unable to correctly assemble the Pdu MCP grow substantially faster than the wild type on 1,2-PD minimal medium with limiting B 12 (49). This phenotype, which is due to increased access of the MCP lumen enzymes to their substrates, is a reliable test of the integrity/permeability of the Pdu MCP (49,50,58). The deletion mutants described above showed a range of growth rates on 1,2-PD at limiting B 12 (Fig.…”

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The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

2017

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Bacterial microcompartments (MCPs) are extremely large proteinaceous organelles that consist of an enzymatic core encapsulated within a complex protein shell. A key question in MCP biology is the nature of the interactions that guide the assembly of thousands of protein subunits into a well-ordered metabolic compartment. In this report, we show that the N-terminal 37 amino acids of the PduB protein have a critical role in binding the shell of the 1,2-propanediol utilization (Pdu) microcompartment to its enzymatic core. Several mutations were constructed that deleted short regions of the N terminus of PduB. Growth tests indicated that three of these deletions were impaired MCP assembly. Attempts to purify MCPs from these mutants, followed by gel electrophoresis and enzyme assays, indicated that the protein complexes isolated consisted of MCP shells depleted of core enzymes. Electron microscopy substantiated these findings by identifying apparently empty MCP shells but not intact MCPs. Analyses of 13 site-directed mutants indicated that the key region of the N terminus of PduB required for MCP assembly is a putative helix spanning residues 6 to 18. Considering the findings presented here together with prior work, we propose a new model for MCP assembly.IMPORTANCE Bacterial microcompartments consist of metabolic enzymes encapsulated within a protein shell and are widely used to optimize metabolic process. Here, we show that the N-terminal 37 amino acids of the PduB shell protein are essential for assembly of the 1,2-propanediol utilization microcompartment. The results indicate that it plays a key role in binding the outer shell to the enzymatic core. We propose that this interaction might be used to define the relative orientation of the shell with respect to the core. This finding is of fundamental importance to our understanding of microcompartment assembly and may have application to engineering microcompartments as nanobioreactors for chemical production.

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Selective molecular transport through the protein shell of a bacterial microcompartment organelle

Cited by 128 publications

References 36 publications

The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

pH determines the energetic efficiency of the cyanobacterial CO ₂ concentrating mechanism

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

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Selective molecular transport through the protein shell of a bacterial microcompartment organelle

Cited by 128 publications

References 36 publications

The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

pH determines the energetic efficiency of the cyanobacterial CO 2 concentrating mechanism

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

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pH determines the energetic efficiency of the cyanobacterial CO ₂ concentrating mechanism